ABSTRACT Alexander's disease (AxD) is a fatal leukodystrophy caused by mutation in the gene encoding glial fibrillary acidic protein (GFAP). Pathologically, AxD is characterized by reactive gliosis, upregulation of GFAP, and accumulation of Rosenthal fibers. AxD is associated with early and progressive loss of neurons and oligodendrocytes. Understanding how an astrocyte specific genetic defect of a non-essential intermediary filament, can so severely influence neuronal function, is critical to developing therapeutic strategies. We believe that the discovery of a brain-wide interstitial clearance system in CNS - the glymphatic system - driven by astrocytes and dependent upon the astrocytic water channel, AQP4, could begin to address this question. This novel clearance path subserves a function homologous to the lymphatics, which in other organs is essential for the clearance of extracellular proteins. We propose that glymphatic flow comprises an important pathway by which GFAP and its proteolytic products can be cleared, but that the formation of Rosenthal fibers, or the toxic effects of GFAP oligomers, and consequent disruption of astroglial cytoarchitecture in AxD result in the mislocation of AQP4, and the consequent failure of glymphatic flow. This in turn leads to reactive changes in affected astroglia, which further increase the transcription of mutated GFAP in a feed-forward process that accelerates both Rosenthal fiber accumulation, and effectively abrogates glymphatic flow, inexorably extending AxD pathology to all cell types and regions within the affected brain. Aim 1 will be to evaluate the contribution of the glymphatic system to the physiological clearance of soluble GFAP in wild type mice using several alternative approaches, including in vivo 2-photon imaging, ex vivo mapping of fluorescently-tagged GFAP distribution at defined time-points, quantitative measurement of radiolabeled GFAP clearance, microdialysis combined with ELISA detecting of endogenous GFAP, immunohistochemistry and Western blot. Aim 2 will characterize the impact of AxD in Gfap+/R236H mice on glymphatic GFAP clearance and manipulate glymphatic clearance by inducible, astrocyte-specific deletion of AQP4 in juvenile AxD transgenic mice. Aim 3 will test the hypothesis that increasing glymphatic clearance in Gfap+/R236H mice by inhibition of inducible nitric oxide synthase (iNOS) will reduce Rosenthal fiber burden and delay age-related impairment of cognitive function. To our knowledge, these studies represent the first analysis of GFAP clearance on an organ level and the first systematic analysis of how AxD affects this clearance pathway. We hope that the experiments will provide fundamentally new insights into the role of glymphatic GFAP clearance on reactive gliosis and Rosenthal fiber burden and thereby define novel targets for treatment of this grave disease.